The viral envelope glycoprotein gpl20 is responsible for the initial events in HIV-1 infection. In its unliganded state, gpl20 is characterized by the presence of intrinsically disordered domains. In particular, the residues that define the co-receptor binding epitope are disordered and only become binding competent when CD4 binds gp 120. The rational design and optimization of inhibitors require a precise knowledge ofthe contribution of each residue at the gp120/CD4 interface to the binding affinity and to the allosteric structuring process. These issues are addressed in terms of the following specific aims: 1. Thermodynamic characterization of the structural stability and cooperative linkage between binding sites in trimeric (solution and membrane bound) and monomeric gp 120 by utilizing a combination of microcalorimetric and structure-based thermodynamic studies. How do intersubunit interactions within the trimer modulate the functional behavior of gp 120? 2. Thermodynamic characterization of conformational changes associated with receptor, co-receptor and inhibitor binding in trimeric (solution and membrane bound) gp 120. These studies will utilize differential scanning calorimetry, isothermal titration calorimetry and spectroscopic probes. 3. CD4 binding allosterically activates gp 120 by triggering a major conformational change. What are the binding hotspots within the CD4/gp120 binding footprint? Do they overlap with the residues that initiate the allosteric cascade? Themodynamic-based alanine scanning mutagenesis will be used to identify binding and allosteric hotspots. Can we inhibit gp120 binding without triggering the allosteric activation cascade? Are the binding and allosteric hotspots the same in the monomer and trimer? Does the membrane affect the interactions? This fundamental information will drive the computational and synthetic components of the POI. 4. Development of thermodynamic guidelines for the design of highly potent, energy efficient gp 120 inhibitors. The thermodynamic signature of all inhibitor candidates will be determined calorimetrically as it captures the essential properties of inhibitors and can be used for further inhibitor optimization. 5. Development of thermodynamic guidelines for gp120 inhibitors that are effective against different HIV-1 subtypes and exhibit low susceptibility to potential drug resistant mutants.